PROJECT SUMMARY/ABSTRACT Functional gastrointestinal diseases (FGIDs), like irritable bowel syndrome, affect ~15% of the US population. Disruptions in the sensation of forces, also known as mechanosensation, are frequent in patients with FGIDs. Therefore, my laboratory?s long-term goal is to elucidate the cellular and molecular mechanisms of gastrointestinal (GI) mechanosensitivity in health and FGIDs. There are several mechanosensory pathways in the GI tract. One important mechanosensory pathway involved in FGIDs is neuro-epithelial, which is composed of a specialized sensory epithelial enteroendocrine cells (EECs) and intrinsic or extrinsic afferent neurons. We discovered a sub-population EECs which are mechanosensitive and express a mechanosensitive ion channel, Piezo2. In these mechanosensitive EECs, force-driven activation of Piezo2 channels is necessary for generation of ?receptor currents? that lead to intracellular Ca2+ increases, the release of signaling molecules and downstream physiologic effects, like epithelial secretion. Thus, Piezo2 EECs appear to be important epithelial mechanosensors, but knowledge gaps limit our ability to target them. The overall objective of this proposal is to determine Piezo2 EECs roles in GI physiology by testing a novel hypothesis that mechanosensitive Piezo2 EECs use a Ca2+ signaling cascade to link Piezo2 activation with release of 5- HT and/or GLP-1 and thereby regulate GI motility and secretion. We will test the hypothesis in 3 Specific Aims. In Aim 1, we will determine the precise mechanotransduction mechanism that connects a very rapid Piezo2 receptor current with prolonged intracellular Ca2+ increase that is necessary for the release of signaling molecules. In Aim 2, we will determine Piezo2 EEC sub-populations based on GI region and the signaling molecules they contain and release. In Aim 3, we will determine how mechanosensitive Piezo2 EECs regulate mechanically induced GI secretion and contractions. We established novel transgenic mouse models that allow us to lineage track, stimulate, and interrogate specific EEC sub-populations. We will use these mouse models and validated EEC lines in a range of innovative and established approaches from single cells to in vivo to determine mechanosensitive EEC functions and their roles in GI physiology. The experiments are foundationally linked to previous work but represent a new and exciting direction and can we can complete in the defined award period. The results from these studies are poised to provide significant advances in the understanding of basic cellular and molecular mechanotransduction mechanisms, sensory epithelial function, GI mechanobiology, and have a broad translational value in physiology. A deep understanding of EEC mechanotransduction positions us well to determine alterations in mechanosensitive EECs in FGIDs, so that we may target them as novel and specific therapies.